Manipulating ATP supply improves in situ CO₂ recycling by reductive TCA cycle in engineered Escherichia coli

The reductive tricarboxylic acid (rTCA) cycle was reconstructed in Escherichia coli by introducing pGETS118KAFS, where kor (encodes α-ketoglutarate:ferredoxin oxidoreductase), acl (encodes ATP-dependent citrate lyase), frd (encodes fumarate reductase), and sdh (encodes succinate dehydrogenase) were tandemly conjugated by the ordered gene assembly in Bacillus subtilis (OGAB). E. coli MZLF (E. coli BL21(DE3) Δzwf, Δldh, Δfrd) was employed so that the C-2/C-1 [(ethanol + acetate)/(formate + CO₂)] ratio can be used to investigate the effectiveness of the recombinant rTCA for in situ CO₂ recycling. It has been shown that supplying ATP through the energy pump (the EP), where formate donates electron to nitrate to form ATP, elevates the C-2/C-1 ratio from 1.03 ± 0.00 to 1.49 ± 0.02. Similarly, when ATP production is increased by the introduction of the heterologous ethanol production pathway (pLOI295), the C-2/C-1 ratio further increased to 1.79 ± 0.02. In summary, the ATP supply is a rate-limiting step for in situ CO₂ recycling by the recombinant rTCA cycle. The decrease in C-1 is significant, but the destination of those recycled C-1 is yet to be determined.